Genetic reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs) by over-expression of specific genes has been accomplished using mouse and human cells. The resulting iPSCs are isogenic to the donor individual, i.e., they carry a similar genetic background, and are thus attractive not only for future therapeutic purposes, with lower risk of immune rejection, but also for understanding complex diseases with heritable and sporadic conditions. However, there are several obstacles to overcome before iPSCs might be considered for cellular therapy; for example, the use of oncogenes and insertional mutagenesis by delivery viruses may induce malignant cell transformation.
Genetic reprogramming to a pluripotent state of mouse somatic differentiated cells was first achieved by ectopic expression of four factors (Oct4, Sox2, Klf4 and c-Myc) using retroviruses (Takahashi, K. & Yamanaka, S., Cell, 2006). Such cells were named induced pluripotent stem cells (iPSCs). Subsequently, the method was applied to human cells using the same factors or a different combination in a lentivirus vector (Oct4, Sox2, Lin28 and Nanog) (Takahashi, K. et al., Cell, 2007; Yu, J. et al., Science, 2007; Lowry, W. E. et al., Proc Natl Acad Sci USA, 2008; Park, I. H. et al., Nature protocols, 2008). Both mouse and human iPSCs seem similar to embryonic stem cells (ESCs) with respect to their morphology, cell behavior, gene expression, epigenetic status and differentiation potential in culture. However, insertional mutagenesis generated by the use of retroviruses increases the risk of tumorigenicity, precluding subsequent safe cellular transplantation (Kustikova, O. et al., Science, 2005). Viral vectors are also known to induce a transcriptional response from target cells, altering their behavior and sometimes inducing apoptosis (Best, S. M., Annu Rev Microbiol, 2008). Moreover, reactivation of viral transgene was also implicated in tumorigenesis from iPSC-derived chimeric mice (Nakagawa, M. et al., Nat Biotechnol, 2008). Finally, random integration may influence the molecular signatures of iPSCs by interrupting regulatory regions in the human genome. iPSCs from mouse fibroblasts were generated using multiple adenoviral infections at an extremely low efficiency (Stadtfeld, M. et al., Science, 2008). A problem with adenovirus mediated transfections is that adenoviral genes may integrate in the genome and induce an immune reaction in humans, raising safety concerns for future therapeutic applications. Recently, iPSCs were generated by transient expression of Oct4, Sox2 and Klf4 from mouse embryonic fibroblasts (Okita, K. et al., Science, 2008). Also, a two-step seamless factor removal from iPS using transposase-stimulated excision was recently reported (Kaji, K. et al., Nature 2009; Woltjen, K et al., Nature 2009). However, these studies need further validation in more rigorous pluripotent assays.
The methods and compositions described herein overcome these and other problems in the art.